1. Field of the Invention
This invention relates to a tissue culture method for abundant production of plant material rich in secondary metabolites, particularly alkaloids of principal interest is a method for culturing poppy plants useful for yielding high levels of morphinan alkaloids, especially thebaine.
2. Description of the Prior Art
The advantages of applying tissue culture to the production of plants rich in secondary metabolites having economic importance are multi-fold. In tissue culture, germination, growth and harvesting are not dependent on season, climate or other specific environmental growth conditions. Moreover, in tissue culture, plants are more readily protected from insects, disease, and adverse weather phenomena.
M. Roberts [Cell Culture and Somatic Cell Genetics of Plants, Vol. 5, (1988) in Academic Press Inc., pp. 315-334] reviews the role of tissue culture in the production of isoquinolines (Papaver alkaloids). This review notes that Papaver species produce a wide range of isoquinolines, and within the individual species there is considerable intraspecific variation in alkaloid content [Phillipson, J. D. (1983) Planta Medica 48:187-192]. Roberts points to P. somniferum L. and P. bracteatum Lindl as the major producers of morphinans (thebaine, codeine and morphine), the most important group of isoquinolines from a commercial and pharmaceutical standpoint. These opiates have been difficult to produce in plant cell cultures.
Lorenz, R. L. et al. [Enzyme Microb. Technol. (1988), 10:219-226] teach that, while the ratio of codeine to morphine can vary, the latex of most P. somniferum varieties will always contain both alkaloids. It is therefore not possible to produce codeine without creating a supply of morphine, most of which enters the illicit drug trade as heroin. Lorenz et al. additionally notes that thebaine, an intermediate on the biosynthetic pathway to codeine and morphine, has become important because it is not addictive and can easily be converted to codeine.
Kamo, K. K. et al. [Phytochemistry 21:219-222 (1982)] explored the influence of the natural plant hormones indoleacetic acid (IAA), isopentenyl adenine (IPA) or the synthetic hormones kinetin (K) and naphthaleneacidic acid (NAA) on the capacity of P. somniferum callus tissues, meristemoids, and redifferentiated roots and shoots to synthesize morphinane alkaloids on artificial media. Kamo et al. found that alkaloid synthesis in tissues appeared to be related to the growth rate of tissues. Fast-growing tissues, such as those grown on IPA or K, had relatively low alkaloid concentrations compared to slow-growing tissues cultured on combinations of K and NAA, or IPA and IAA. Also, shoot organs redifferentiated on callus produced greater quantities of morphinane alkaloids than the callus, itself, though the level of production was less than for shoots of intact seedlings.
Schuchmann, R. et al. [Plant Cell Reports (1983) 2:88-91] reports that the alkaloid patterns of in vitro regenerated plantlets were at least qualitatively similar to those of normally grown seedlings. Thebaine accumulated up to 0.2% by weight of dry matter. In normally-grown seedlings of the same size, thebaine levels were 0.03% (7-day) and 0.01% (26-day).
Kutchan, T. M. et al. [Plant Cell Reports (1983) 2:281-284] extracted thebaine from P. bracteatum green shoots and meristemoids in static culture at levels that were comparable to some of the highest levels previously reported. These results were obtained by initially growing the tissues in the presence of 5 ppm 6-benzylaminopurine (BA) or 2 ppm indolebutyric acid (IBA) and then transferring them to media without 2,4-dichlorophenoxyacetic acid (2,4-D), BA or IBA. The levels of thebaine were still generally three orders of magnitude lower than amounts in latex-containing mature flower stems.
In a study focused on the effects of photoperiod and temperature on in vitro regeneration of plantlets from callus cultures of P. somniferum, Yoshikawa, T. et al. [Experientia (1983) 39:1031-1033] observed that callus was actively promoted at 16-18° C., but inhibited at >25° C. Shoots grown at 16-18° C. grew to 10-15 cm and leaves appeared normal.
Day, K. B. et al. [Plant Cell Reports (1986) 5:471-474] observed that plants regenerated from embryogenic callus cultures of two varieties of P. bracteatum and transplanted to soil yielded thebaine concentrations comparable to those in seed-grown plants. Green shoots up to 8 mm long were produced from some of the white meristemoid regions within 3-4 weeks of transfer to solidified Murahige and Skoog (MS) medium. Thebaine levels of plantlets cultivated on MS medium remained modest, but increased significantly when the plantlets were transferred to soil and grown in the greenhouse. The leaves of one particular plant produced 517 μg thebaine/g fresh weight after 5 months on MS plus 5 months in the greenhouse.
Kaseem, M. A. et al. [J. Biomedicine and Biotechnology 1:2 (2001) 70-78] observed that stems and leaves or explants from seed-grown P. somniferum album contained morphine (0.023%), codeine (0.013%) and thebaine (0.017%). Similar results were obtained with P. somniferum. 